WO2017197588A1

WO2017197588A1 - Glyphosate-resistant gene screening method, epsps mutant gene and deficient strain and use

Info

Publication number: WO2017197588A1
Application number: PCT/CN2016/082409
Authority: WO
Inventors: 王博雅; 卢远根; 胥南飞
Original assignee: 四川天豫兴禾生物科技有限公司
Priority date: 2016-05-17
Filing date: 2016-05-17
Publication date: 2017-11-23
Also published as: US20190144882A1; US10961546B2; CA3023994A1; CA3023994C

Abstract

Provided are a glyphosate-resistant gene screening method, an EPSPS mutant gene having glyphosate resistance screened by the method, an EPSPS and C-P Lyase deficient strain and a use thereof.

Description

Glyphosate resistant gene screening method, EPSPS mutant gene and defective strain and application thereof

Technical field

The present invention relates to the field of biotechnology, and in particular to a glyphosate resistant gene screening method, an EPSPS mutant gene, and a defective strain and application.

Background technique

Developed by Monsanto, USA, Glyphosate is a foliar spray, broad-spectrum, non-selective systemic glyphosate. The main component of glyphosate is to inhibit the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway in plants, so that the affected plants can not continuously synthesize essential amino acids and affect the normal growth of plants. Even death.

Glyphosate is a broad-spectrum glyphosate herbicide that is damaging to almost all plants. The most commonly used glyphosate-resistant gene on the market is the CP4 gene, which is a strong resistance to glyphosate isolated from Agrobacterium. Gene. Plants can acquire this resistance by means of transgenesis. Because glyphosate-resistant crops bring significant benefits to agriculture and the environment, corn and soybean varieties containing the CP4 gene have been widely promoted in the past 20 years. However, new glyphosate resistant genes and glyphosate resistant varieties based on this are still in need in production applications.

It is best to use genetic editing techniques to create non-transgenic glyphosate resistant plant EPSPS genes that are resistant to glyphosate. Although the microbial EPSPS gene represented by CP4 can provide glyphosate resistance, even if these genes are genetically modified into plants, they are still suspected of transgenic because of different species, and it is difficult to obtain recognition from the general public. The public's understanding of GM crops here is biased, which hinders the development and popularization of GM technology to some extent. Therefore, creating a plant glyphosate-resistant EPSPS gene is a key to non-GMO-resistant glyphosate crops.

In theory, the plant's own EPSPS gene can be mutagenized by chemical, radiation or other methods to screen resistant plants under certain glyphosate pressures. In fact, some weeds have evolved resistance to glyphosate in the case of extensive use of glyphosate for many years; most of them are changes in the EPSPS gene. But these changes are mostly the increase in gene copy number, and the resistance is not very high, it is difficult to use on crops. The EPSPS gene of the crop itself also has examples of mutations that produce glyphosate resistance, but the resistance is not as good as CP4. In order to create non-GM crops that are highly resistant to glyphosate, it is imperative to continue to mutagenize and screen crops or other plant EPSPS gene resistance genes.

However, the existing method for screening a mutant gene having glyphosate resistance from crops or other plants requires first mutagenizing the plants to obtain a large number of mutant plants, and then screening the mutant plants for resistance. A mutant plant having glyphosate resistance is finally analyzed to obtain a glyphosate-resistant mutant gene by genomic detection analysis of the resistant plant. Due to the long period of plant growth, planting a large number of mutant plants not only takes a long time and requires a large land area.

Summary of the invention

The object of the present invention is to provide a screening method for a mutant gene having glyphosate resistance, which is capable of rapidly screening a mutant gene derived from a plant from a foreign gene, and the mutant gene screened by the screening method has a glyphosate Phosphine resistant.

A further object of the present invention is to provide a mutant gene which is screened by the above screening method and which has glyphosate resistance.

Still another object of the present invention is to provide an application of the above mutant gene such that a plant transformed with the mutant gene has glyphosate resistance.

Still another object of the present invention is to provide a model strain for screening a mutant gene having glyphosate resistance, which does not express EPSPS nor has a function of lysing glyphosate.

Still another object of the present invention is to provide the use of the above model bacteria for testing the function of a plant-derived EPSPS gene.

Still another object of the present invention is to provide the use of the above model bacteria for testing the resistance of a plant-derived EPSPS gene to glyphosate.

A further object of the present invention is to provide the use of the above model bacteria for testing the resistance of a plant-derived mutant EPSPS gene to glyphosate.

The technical problem solved by the present invention is achieved by the following technical solutions.

A method for screening glyphosate resistant genes, comprising:

The gene was knocked out to knock out the interfering gene of the source strain, and the defective strain was obtained. The source strain was from one of Escherichia coli DH5α, TOP10 and BL21. The interference genes included EPSPS gene and CP Lyase gene, and the defective strains were EPSPS and CP. Lyase-deficient strain;

The exogenous EPSPS gene was first introduced into the defective strain, and then the mutagenized treatment was carried out to obtain the first mutant strain containing the exogenous EPSPS mutant gene, and the exogenous EPSPS gene was derived from the target plant;

Alternatively, the exogenous EPSPS gene is firstly mutated to obtain an exogenous EPSPS mutant gene, and the exogenous EPSPS mutant gene is introduced into the defective strain to obtain a second mutant strain;

The first mutant strain or the second mutant strain is placed on a screening medium containing glyphosate for screening culture to obtain a monoclonal resistant strain having glyphosate resistance;

The monoclonal resistant bacteria were sequenced to obtain an EPSPS mutant gene with glyphosate resistance.

The beneficial effects of the glyphosate resistant gene screening method, the EPSPS mutant gene and the defective strain provided by the present invention and the application thereof are: the present invention, compared with the prior screening method for screening a mutant gene having glyphosate resistance from plants The screening method was constructed by constructing EPSPS and CP Lyase-deficient strains, and then using the EPSPS and CP Lyase-deficient strains as host bacteria, introducing the exogenous EPSPS gene from the plant of interest into the defective strain, and obtaining the exogenous EPSPS mutation. The mutant strain of the gene is the exogenous EPSPS gene mutant library, and the EPSPS mutant gene with glyphosate resistance is screened from the exogenous EPSPS gene mutant library. Because the bacteria are fast in reproduction and small in volume, the screening method of the present invention overcomes the problems of long cycle and large area in the existing screening methods, so that the screening method of the present invention screens glyphosate in a targeted manner. The resistant EPSPS gene has a short cycle, a small footprint, a short cycle and simple operation. Moreover, the screening method of the present invention uses EPSPS and CP Lyase-deficient strains as host bacteria, and effectively excretes the EPSPS gene of the host bacteria itself and the mutation of the CP Lyase gene to produce glyphosate resistance, so that the selected results are obtained. More scientific and more reliable.

DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly described below. It should be understood that the following drawings show only certain embodiments of the present invention, and therefore It should be seen as a limitation on the scope, and those skilled in the art can obtain other related drawings according to these drawings without any creative work.

1 is a structural diagram of a pADV5 carrier according to an embodiment of the present invention;

2 is a structural diagram of a pKD46 carrier according to an embodiment of the present invention;

3 is a result of sequence comparison analysis of a rice EPSPS mutant gene and a wild type rice EPSPS gene according to Example 1 of the present invention;

4 is a result of sequence comparison analysis of soybean EPSPS mutant gene and wild type soybean EPSPS gene according to Example 2 of the present invention.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in order to clarify the objects, the technical solutions and the advantages of the embodiments of the present invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.

The glyphosate resistance gene screening method, the EPSPS mutant gene, and the defective strain and application of the present invention are specifically described below.

A method for screening glyphosate resistant genes, comprising:

Step S1: constructing a defective strain

The gene was knocked out to knock out the interfering gene of the source strain, and the defective strain was obtained. The source strain was from one of Escherichia coli DH5α, TOP10 and BL21. The interference genes included EPSPS gene and CP Lyase gene, and the defective strains were EPSPS and CP. Lyase-deficient strain.

That is, the EPSPS and C-P Lyase-deficient strains are defective strains obtained by knocking out the EPSPS gene and the C-P Lyase gene in one of Escherichia coli DH5α, TOP10, and BL21. The EPSPS and CP Lyase-deficient strains are characterized by the inability to grow in a basal medium containing no amino acids or proteins, which can also be referred to as a limiting medium, but can be based on a sugar-only basis after introduction of the exogenous EPSPS gene. Growing on the medium.

The action of knocking out the source strain, that is, knocking out the EPSPS gene and the C-P Lyase gene of wild-type Escherichia coli, is as follows.

Since the endogenous EPSPS gene of E. coli can express EPSPS5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and the CP Lyase gene can also express CP lyase that cleaves the CP bond, the CP lyase can cleave the grass. Glyphosate. Therefore, if the wild type Escherichia coli is used as the host strain, the EPSPS gene and the CP Lyase gene endogenous to the host strain may also be mutated in the subsequent mutagenesis treatment step, resulting in an EPSPS mutant gene endogenous to glyphosate resistant. And the lytic-enhanced CP Lyase mutant gene confers glyphosate resistance to the host strain, resulting in the inability to clearly identify whether the glyphosate resistance of the monoclonal resistant strain is conferred by the exogenous EPSP mutant gene or its endogenous EPSPS. The mutated gene and the CP Lyase mutant gene are conferred. Therefore, when Escherichia coli is used as the host strain, it is necessary to knock out the endogenous EPSPS gene and CP Lyase gene to ensure that the glyphosate resistance of the finally obtained monoclonal resistant strain is conferred by the exogenous EPSPS mutant gene. To make the results of the screening more scientific, more reasonable and more reliable.

Of course, there are many knock-out techniques for knocking out the EPSPS gene and the C-P Lyase gene of Escherichia coli, such as the FRT method, the pCas system, the use of the pKD46 system, or the direct knockout using homologous PCR fragments. In the case where the E. coli genome sequence information is clear, the EPSPS gene and the C-P Lyase gene on the genome can be easily knocked out by the above methods.

Step S2: Constructing a library of exogenous EPSPS gene mutants

One construction strategy is to use the defective strain as a host strain, first introduce the exogenous EPSPS gene into the defective strain, and then obtain the first mutant strain containing the exogenous EPSPS mutant gene after the mutagenesis treatment. Preferably, the mutagenesis treatment is a chemical mutagenesis treatment or a radiation mutagenesis treatment. The chemical mutagenesis treatment uses, for example, a chemical mutagen such as EMS or DES to mutagenize the first mutant strain so that the exogenous EPSPS gene is mutated with the proliferation of the host strain.

Another construction strategy is to first mutate the exogenous EPSPS gene to obtain the exogenous EPSPS mutant gene, and then introduce the exogenous EPSPS mutant gene into the defective strain to obtain the second mutant strain. Among them, the mutation treatment is a PCR product obtained by PCR using a mismatch PCR method or a DNA Shuffling method as an exogenous EPSPS gene as a template, which is an exogenous EPSPS mutant gene.

It should be noted that the first mutant strain or the second mutant strain all contain an exogenous EPSPS mutant gene, and the first mutant strain or the second mutant strain are all exogenous EPSPS gene mutant libraries.

The terms "first" and "second" are used merely to distinguish the purpose of the description, and are not to be construed as indicating or implying relative importance.

The exogenous EPSPS gene used in the above steps is derived from the plant of interest, and the target plants are rice, soybean, wheat, corn, barley, sorghum, tobacco, cotton, sweet potato, poplar, potato, cabbage, kale or green pepper. In the actual screening process, it can be selected according to actual needs.

Step S3: Resistance screening

The first mutant strain or the second mutant strain obtained from the exogenous EPSPS gene mutant library obtained in step S2 is subjected to screening culture on a screening medium containing glyphosate to obtain a monoclonal resistant strain having glyphosate resistance. . It should be noted that the monoclonal resistant bacteria, that is, the colonies that grow on the screening medium, may also be referred to as positive transformants. Of course, there are many cases of the number of positive transformants, such as one positive transformant or multiple positive transformants.

Among them, the screening medium is M9 basic medium containing different glyphosate concentrations.

Step S4: Sequencing verification

The monoclonal resistant strain obtained in the step S3 was subjected to sequencing verification to obtain an EPSPS mutant gene having glyphosate resistance.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

In this example, the target plant is rice (Oryza sativa), the exogenous EPSPS gene is rice EPSPS gene (the nucleotide sequence thereof is shown in SEQ ID NO. 1), and the wild type is knocked out by direct knockout using homologous PCR fragments. The EPSPS and CP Lyase-deficient strains obtained from the EPSPS gene and the CP Lyase gene in Escherichia coli DH5α are examples of host bacteria, and the screening method of the present invention is described in more detail. The primer names and nucleotides used in the present example are used. The sequence is shown in Table 1.

In the first step, the EPSPS gene and the C-P Lyase gene in E. coli DH5α were directly knocked out using a homologous PCR fragment.

1. Knock out the C-P Lyase gene of Escherichia coli DH5α

(1) homologous PCR fragment amplification

Using the forward primer CPF2, the reverse primer CP5HA3 (see Table 1), the wild type Escherichia coli DH5α was used as a template for PCR, and the gel was recovered to obtain a PCR product, which was named CP5HA fragment, and the length was 525 bp, and the nucleotide sequence thereof was as follows. SEQ ID NO. 13 is shown.

Table 1. Primers used in this example and their nucleotide sequences

PCR was carried out using the forward primer with CP3HA5 as the reverse primer CPR2 and Escherichia coli DH5α as template. The PCR product was obtained and the CP3HA fragment was named with a length of 503 bp. The nucleotide sequence is shown in SEQ ID NO. .

Using the forward primer SPEC5, the reverse primer SPEC3, the vector containing the nucleotide sequence shown in SEQ ID NO. 2 (the vector was named pCPSG7) was used as a template for PCR, gel recovery, and a PCR fragment was obtained, which was named SPEC. The fragment has a length of 900 bp and its nucleotide sequence is shown in SEQ ID NO.

Using CPF2 and CPR2 as primers, PCR was carried out using CP5HA fragment, SPEC fragment and CP3HA fragment as template (in the same reaction system), and the gel was recovered to obtain a PCR product named CP5HA-SPEC-CP3HA fragment with a length of 1849 bp. The nucleotide sequence is shown in SEQ ID NO. Among them, the first to the 525th are the 5th end of the PhneA gene of Escherichia coli and its upstream sequence, and the nucleotide sequence of the 526th to the 1346th is the Spectinomycin resistance gene and its promoter, and the 1347th to the 1849th It is the 3 end of the Escherichia coli PhnH gene and its downstream sequence.

(2) hot hit conversion

Escherichia coli DH5α competent cells were prepared in a conventional manner. 100 μL of E. coli DH5α competent cells were gently mixed with 5 μL of CP5HA-SPEC-CP3HA fragment, placed on ice for 10 min, heat-shocked at 42 °C for 90 s, and immediately transferred to ice for 2 min.

Rapidly add 1 mL of LB liquid medium (Spec (spectinomycin, spectinomycin) containing 50 μg/mL), and incubate at 37 ° C for 1 hr, then apply to LB solid medium (Spec with 50 μg / mL Spec), at 37 Incubate overnight at °C.

After the cultured Escherichia coli DH5α was detected by the forward primer SPE35 and the reverse primer CPR0, the strain was named EDC, and EDC was Escherichia coli DH5α which knocked out the C-P Lyase gene.

2. Eps the EPSPS gene of EDC (E. coli DH5α knockout C-P Lyase gene)

(1) homologous PCR fragment amplification

The wild type Escherichia coli DH5α was used as a template, and the forward primer EE5-1K and the reverse primer ES5HA3 were used for PCR and gel recovery to obtain a PCR product, which was named ES5HA fragment, and the length was 1194 bp, and the nucleotide sequence thereof was SEQ ID. Shown in NO.17.

Using E. coli DH5α as a template, PCR was carried out with the forward primer ES3HA5 and the reverse primer EE3-1K, and the PCR product was obtained. The PCR product was obtained and named as ES3HA fragment, which was 1168 bp in length and its nucleotide sequence was as SEQ ID NO. Show.

Using the forward primer GM5L and the reverse GM3L, the vector containing the nucleotide sequence shown in SEQ ID NO. 3 (the vector was named pCPSG5) was used as a template for PCR, and the gel was recovered to obtain a PCR product, which was named as a GM fragment. The length is 1050 bp, and the nucleotide sequence thereof is shown in SEQ ID NO.

Using EE5-1K and EE3-1K as primers, ES5HA fragment, GM fragment and ES3HA fragment as template to carry out PCR and gel recovery, and obtain PCR product, named ES5HA-GM-ES3HA fragment, the length is 3322 bp, and its nucleotide sequence is as follows. SEQ ID NO. 20 is shown. Among them, the first to the 1194th are the upstream sequence of the E. coli EPSPS gene, and the nucleotide sequences from the 1195th to the 2154th are the gentamicin resistance gene and its promoter, and the 2155th to the 3322th are The downstream sequence of the E. coli EPSPS gene.

(2) hot hit conversion

EDC competent cells were prepared in a conventional manner. 100 μL of LEDC competent cells were gently mixed with 5 μL of ES5HA-GM-ES3HA fragment, placed on ice for 10 min, heat-shocked at 42 °C for 90 s, immediately transferred to ice for 2 min; rapidly added 1 mL of LB liquid medium, and incubated at 37 ° C for 1 hr. The cloth was incubated on an LB solid medium (containing 50 μg/ml Spec and 50 μg/ml Gm) containing Spec (50 μg/ml) and Gm (50 μg/ml), and cultured overnight at 37 °C.

The cultured strain was detected with the forward primers EE5-1K and GM3L, the reverse primers EE3-1K and ECES35U, and the strain was named EDCE. EDCE is Escherichia coli DH5α, which is an EPSPS and C-P Lyase-deficient strain, after knocking out the EPSPS gene and the C-P Lyase gene.

Of course, other conventional knockout methods can be used, for example, to knock out the EPSPS gene and the C-P Lyase gene in E. coli DH5α using the pCas system, or to knock out the EPSPS gene and the C-P Lyase gene in E. coli DH5α using the pKD46 system.

In the second step, the EPSPS and C-P Lyase-deficient strains obtained in the first step are used as host bacteria, and the EPSPS gene derived from rice is introduced into the host strain to obtain a mutant strain, that is, a rice EPSPS gene mutant library. The specific operation is as follows.

1. Construction of EPSP gene mutant library by mismatch PCR

The mRNA of the rice EPSPS gene was reverse transcribed into cDNA by a conventional method and cloned into the pADV5 vector (the structure is shown in Fig. 1).

The first round of mismatch PCR was performed using the forward primer PV325 and the reverse primer PV323 with the pADV5 vector template linked to the rice EPSPS gene. The PCR reaction system consisted of 25.3 μL of H ₂ O, 4 μL of error-prone PCR MIX, and 4 μL. Error-prone PCR dNTPs, 4 μL of MnCl ₂ , 0.8 μL of PV325, 0.8 μL of PV323, 0.1 μL of Taq enzyme, 2 μL of template. The PCR reaction procedure: 95 ° C, 30 seconds; 60 ° C, 30 seconds; 72 ° C, 2 minutes; 40 cycles of PCR products were electrophoresed on 1% agarose, and then the gel was recovered to obtain the first round of PCR product.

The second round of PCR was carried out using the first round of PCR product as a template and the forward primer 2M1H and the reverse primer 2M1T. The PCR system was: 31.9 μL of H ₂ O, 2.5 μL of DMSO, 5 μL of 10× PCR buffer, 5 μL of dNTP, 4 μL of MgCl ₂ , 0.5 μL of 2M1H, 0.5 μL of 2M1T, 0.1 μL of Taq, 0.5 μL of template. . The PCR reaction procedure: 95 ° C, 30 seconds; 60 ° C, 30 seconds; 72 ° C, 2 minutes; 60 cycles

The obtained PCR product was subjected to 1% agarose electrophoresis, and the band of the target band size (1.5 kb) was subjected to gel recovery and purification, and the purified product was subjected to double digestion with Pac1 and Sbf1, and then ligated to the same double digestion. After the new pADV5 vector, the ligation product was obtained. This step resulted in the ligation product being the pADV5 vector carrying the rice EPSPS mutant gene.

Of course, the pADV5 vector carrying the rice EPSPS mutant gene can also be obtained by the DNA Shuffling method, and the specific operation is as follows.

The DNA shuffling method was used to obtain the pADV5 vector carrying the rice EPSPS gene mutant: 1 PCR amplification was carried out with the rice EPSPS gene sequence, and the amplified product was electrophoresed with 1% agarose, then recovered by gel; 2 pairs of recovered products were treated with DNase Digestion, after digestion, run 1.2% agarose electrophoresis, cut 100bp, 200bp or 300bp size fragments for gel recovery and purification; 3 use the gel recovery product 3μL in the second step as a template for gene shuffling first round PCR, In this round of PCR, no primers were added and 60 cycles were amplified. 4 10 μL of the 3rd step PCR product was run and electrophoresed to see if there was a continuous range of large fragments. If expected, the remaining PCR products were used as templates for the next round of PCR. 5 Take the PCR product of step 3, 0.5 μL, for the next round of PCR. This round of PCR primers is designed to have primers with restriction sites, and 60 cycles are amplified. 6 Take the 5th step PCR product. 1% agarose gel electrophoresis, cut a single band larger than 500 bp for gel recovery and purification; 7 double-enzyme digestion of the gel recovery product with restriction enzymes, double digestion and 1% agarose gel Electrophoresis, cut the purpose Segment, with the glue squeeze frozen in liquid nitrogen, and then connected to the carrier pADV5 same double digestion. A plurality of pADV5 vectors carrying a gene mutant of the rice EPSPS gene were obtained.

(2) Transformation of EDCE (E. coli DH5α after knockout of EPSPS gene and C-P Lyase gene)

EDCE competent cells were prepared in a conventional manner. The above ligated product (pADV5 vector carrying rice EPSPS mutant gene) was added to 50 μL of EDCE competent cells, mixed well on ice for 30 min; heat shocked at 42 °C for 90 s, and added to LB liquid medium 500 μL after 2 min in ice bath; Incubate at a low speed (150 r/min) for 90 min at a low speed.

The pADV5 vector carrying the rice EPSPS mutant gene was transformed into EDCE to obtain a mutant strain, that is, a rice EPSPS gene mutant library. The rice EPSPS gene mutant library contains a large number of rice EPSPS mutant genes. Among them, each mutant is equivalent to a rice EPSPS gene mutant plant. Therefore, if screening the rice EPSPS mutation gene of the same order of magnitude, the screening method of the present invention does not have to go through the rice culture period and the occupied land area, and the time, efficiency and operation process are very fast, compared with the existing screening methods. Simple, especially for very small footprints, screening is done only on the medium.

In the third step, the above mutant bacteria are inoculated to a screening medium for resistance screening.

The plurality of mutant strains obtained above were separately inoculated on a plurality of screening mediums containing different concentrations of glyphosate (the screening medium contained a difference in the concentration of glyphosate between them, and the concentrations of glyphosate-containing were respectively 10 mM, 20 mM, 50 mM, etc., glyphosate having a difference in concentration gradient, of course, the concentration of glyphosate can be set according to the actual conditions), and cultured at 37 ° C overnight. Among them, the screening medium is based on M9, and a certain concentration of antibiotics Spec (Spectinomycin, spectinomycin), Gen (Gentamycin, gentamicin), Amp (Ampicillin, ampicillin) and different concentrations are added. The medium obtained from glyphosate. The components of the M9 medium are as follows: Na ₂ HPO ₄ 13 to 14 g/L, KH ₂ PO ₄ 5.7 to 6.3 g/L, NaCl 0.9 to 1.1 g/L, NH ₄ Cl 1.8 to 2.2 g/L, and glucose 37 to 43 g. /L, MgSO ₄ ·7H ₂ O 48 to 52 g/L, and CaCl ₂ 21 to 23 g/L.

The fourth step is sequencing verification.

The monoclonal resistant bacteria grown on the screening medium were selected, isolated, and tested for glyphosate resistance, and sequenced to obtain a sequence of the glyphosate-resistant rice EPSPS mutant gene. One of the rice EPSPS mutant genes is described, and its nucleotide sequence is as shown in SEQ ID NO. 4, and is composed of 1365 bases. The rice EPSPS mutant gene (designated OsEM gene) is compared with the nucleotide sequence of the wild type rice EPSPS gene (designated as OsE gene) (as shown in SEQ ID NO. 1) and its encoded amino acid sequence, The result is shown in Figure 3. The rice EPSPS mutant gene was mutated from "C" to "G" from the 5' to the 3' end, and the 240th base was changed from "T" to "C", 346th and 347th. Two consecutive bases "CT" are mutated to "TC", the 396th base "T" is mutated to "C", the 453th base "A" is mutated to "G", and the 606th base "C" The mutation is "T", and the 833th base "A" is mutated to "G"; wherein only the 209th base is mutated from "C" to "G", resulting in the sequence of the encoded amino acid residue from the amino terminus to The 70th position of the carboxy terminus is mutated from alanine residue to a glycine residue, and the 246th and 347th consecutive two bases are "CT" mutated to "TC", resulting in the 116th position of the encoded amino acid residue sequence. The leucine residue was mutated to a serine residue, and the remaining base mutations did not result in a change in the encoded amino acid residue.

Glyphosate resistance test of rice EPSPS mutant gene was inoculated with Escherichia coli (EPSPS and CP Lyase-deficient strain) transformed with OsEM gene (experimental group) and OsE gene (control group) and contained 0 mM, 1 mM, 5 mM, The growth of Escherichia coli was observed in a medium of 10 mM, 20 mM, 50 mM, 10 mM glyphosate (expressed by growth saturation index, saturation index = 0, no growth; saturation index = 1, small growth; saturation index = 2) , to semi-saturated; saturation index = 3, vigorous growth, but there is room for growth; saturation index = 4, rapid growth, bacteria have reached the highest (saturated) concentration in the medium or growth has reached the limit). The results are shown in Table 2.

Table 2. Growth saturation index of Escherichia coli transformed with OsEM gene and OsE gene in glyphosate medium containing different concentrations

As can be seen from Table 2, the experimental group (containing OsEM gene) and the control group (containing OsE gene) were able to grow normally on the medium containing 0 mM monoglyphosate (saturation index was 4); in 1 mM, 5 mM, 10 mM, On the medium of 20 mM, 50 mM glyphosate, the control group could not grow (saturation index is 0), while the experimental group can grow normally (saturation index is 4); on the medium containing 500 mM glyphosate, the experimental group and the control None of the groups grew normally (saturation index was 0). This indicates that the rice EPSPS mutant gene (the nucleotide sequence of which is shown in SEQ ID NO. 4) screened in this example can confer E. coli glyphosate resistance to EPSPS and CP Lyase-deficient Escherichia coli. Growth was carried out in a medium of 50 mM glyphosate.

The glyphosate-resistant mutant gene, such as the rice EPSPS mutant gene, screened by the glyphosate-resistant gene screening method provided by the present invention, has a nucleotide sequence as shown in SEQ ID NO. 4, which has Resistance to 50 mM glyphosate.

The glyphosate-resistant mutant gene, such as the rice EPSPS mutant gene (the nucleotide sequence thereof is shown in SEQ ID NO. 4), which is screened by the glyphosate resistance screening method provided by the present invention, is directly transformed. Rice or soybean or other plant to make the transformed plant have glyphosate resistance. Of course, transformation methods commonly used in the field of genetic engineering, such as Agrobacterium-mediated methods, The rice plant or soybean or other plant is transformed by a gene gun method, a protoplast-mediated method, an electric shock method or a whole-level transformation method to make the transformed plant have glyphosate resistance.

Example 2

In this embodiment, the target plant is soybean (Glycine max), the foreign gene is soybean EPSPS gene (the nucleotide sequence thereof is shown in SEQ ID NO. 5), and the wild type is directly knocked out by homologous FRT method. The EPSPS and CP Lyase-deficient strains obtained from the EPSPS gene and the CP Lyase gene in Escherichia coli DH5α are taken as host bacteria to illustrate the screening method of the present invention. The primer names and nucleotide sequences used in the present embodiment are shown in Table 3. .

In the first step, the C-P Lyase gene of Escherichia coli DH5α was knocked out.

The EPSPS gene and C-P Lyase gene in E. coli DH5α strain were knocked out by FRT method, and knocked out in two steps. The C-P Lyase gene was knocked out first, and then the EPSPS gene was knocked out.

1. Preparation of E. coli DH5α competent cells containing pKD46 plasmid

0.5 μL of pKD46 plasmid (the structure is shown in Figure 2) was transformed into E. coli DH5α competent cells, and positive colonies were screened on LB medium plates (containing Amp100);

The positive monoclonal colonies were picked and inoculated into a small amount of M9-sucrose liquid medium (containing sucrose), and cultured at 180 rpm and 30 ° C overnight;

After the completion of the culture, a large amount of M9-sucrose liquid medium (containing sucrose + 100 μg / mL Amp + 10 mM L-arabinose) was inoculated at a ratio of 1:10, and cultured at 30 ° C until the OD600 of the bacterial liquid was about 0.7 to about 0.7;

The above bacterial solution was cooled on ice for 20 min, and the cells were collected by centrifugation at 4 ° C and 4000 rpm; resuspended in 40 mL of pre-cooled 10% (v/v) glycerol, washed repeatedly for 3 times, and the supernatant was discarded, and pre-cooled with 400 μL. The 10% glycerol was resuspended and dispensed into 100 μL/tube to obtain AH-resistant DH5α.

2. Knock out the C-P Lyase gene of Escherichia coli DH5α

Using the forward primer C-P Lyase_P15 and the reverse primer C-P Lyase_P13 (see Table 3), PCR amplification was carried out using the E. coli DH5α genome as a template to obtain a P1 fragment, and the nucleotide sequence of the P1 fragment is shown in SEQ ID NO.

PCR amplification was carried out using the forward primer C-P Lyase_P25, the reverse primer C-P Lyase_P23, and the E. coli DH5α genome as a template to obtain a P2 fragment. The nucleotide sequence of the P2 fragment is shown in SEQ ID NO.

P1 and P2 were purified by electrophoresis on 1% agarose to obtain a purified PCR product, and a plasmid containing a Gen-resistant fragment was added in proportion to prepare a pool, using the forward primer CP Lyase_P15 and the reverse primer CP Lyase_P23. For PCR amplification, a PRC fragment was obtained, which was 1586 bp in length and its nucleotide sequence is shown in SEQ ID NO.

50 μL of E. coli DH5α competent cells (AH-resistant DH5α) was gently mixed with 30 μL of the purified PRC fragment, placed in a 0.1 cm pre-cooled electric shock cup, and subjected to electric shock at 1.8 kV using a Bio-Rad electrorotator;

1 mL of M9-sucrose liquid medium containing 10 mM arabinose was quickly added, and cultured at 30 ° C for 1 h, and then applied to LB solid medium (100 μg/mL of Amp and 30 μg/mL of Gen) to screen for simultaneous anti-Amp and Gen. Recombinant strain, cultured overnight at 30 ° C;

After the completion of the culture, the positive clone containing the Gen gene was screened with the forward primer C-P Lyase_5UTR and the reverse primer C-P Lyase_Gen3 to verify the presence of the Gen gene;

The positive clone was inoculated into LB+Amp liquid medium, cultured at 30 ° C overnight (12 hr), then transferred to fresh LB liquid medium, and continued to culture at 30 ° C for 12 hr;

The culture solution was diluted to an appropriate concentration, and the LB plate was coated, and the clone without the Gen gene was screened with the forward primer C-P Lyase_5 UTR and the reverse primer Lyase_3 DSR primer;

The monoclonal sequence was picked and the strain was preserved and named DH46ΔC-P Lyase. DH46ΔC-P Lyase is Escherichia coli DH5 which knocks out the C-P Lyase gene.

Table 3. Primer names and nucleotide sequences used in this example

3. Knock out DH46ΔC-P Lyase (E. coli DH5α knockout C-P Lyase gene) EPSPS gene

(1) Preparation of DH46ΔC-P Lyase competent cells

The preserved DH46ΔC-P Lyase was streaked on LB+Amp plate and cultured overnight at 30° C. The positive monoclonal colonies were picked and inoculated into a small amount of M9-sucrose liquid medium and cultured at 180 rpm and 30° C. overnight.

After the completion of the culture, in a ratio of 1:10 in a large amount of M9 liquid medium (containing sucrose + 100 μg / mL Amp + 10 mM L-arabinose), cultured at 30 ° C until the OD600 of the bacterial solution was 0.7;

The above bacterial solution (containing DH46ΔC-P Lyase) was cooled on ice for 20 min, and the cells were collected by centrifugation at 4° C. and 4000 rpm; resuspended in 40 mL of pre-cooled 10% (v/v) glycerin, washed repeatedly for 3 times and discarded. Clear, resuspended in 400 μL of pre-cooled 10% glycerol and dispensed into 100 μL/tube.

(2) homologous PCR fragment amplification

Using the forward primer EcEPSPS_P35 and the reverse primer EcEPSPS_P33, the DH46ΔC-P Lyase strain genomic DNA was used as a template to obtain a product P3 fragment, and the nucleotide sequence of the P3 fragment is shown in SEQ ID NO.

Using the forward primer EcEPSPS_P45 and the reverse primer EcEPSPS_P43, the DH46ΔC-P Lyase genomic DNA was used as a template to obtain a product P4 fragment, and the nucleotide sequence of the P4 fragment is shown in SEQ ID NO.

The P3 fragment and the P4 fragment were purified by 1% agarose electrophoresis, and the plasmid containing the Gen-resistant fragment was added into a mixing pool in proportion, and the template was used to amplify with EcEPSPS_P35 and EcEPSPS_P43 as primers to obtain a product PRE fragment. It is 1607 bp and its nucleotide sequence is shown in SEQ ID NO.

(3) Hot hit conversion

50 μL of DH46ΔC-P Lyase competent cells were gently mixed with 35 μL of the purified PRE fragment, placed in a 0.1 cm pre-cooled electric shock cup, and subjected to electric shock at 1.8 kV using a Bio-Rad electrorotator;

1 mL of M9-sucrose liquid medium containing 10 mM arabinose was quickly added, cultured at 37 ° C for 1 hr, and then plated on LB solid medium to screen recombinant strains, and cultured overnight at 30 ° C; the next day, EcEPSPS_P35 and EcEPSPS_P43 primers were used to screen for containing Gen A positive clone of the gene verified the presence of the Gen gene.

The positive clone was inoculated into LB liquid medium, cultured at 37 ° C overnight (12 hr), then transferred to fresh LB liquid medium, and continued to culture at 37 ° C for 12 hr;

The culture solution was diluted to an appropriate concentration, and the LB plate was coated, and the clone without the GM gene was selected using the forward primer EcES25 and the reverse primer forward primer EcES23;

Monoclonal sequencing was carried out, and the strain was preserved and designated as DH5αΔPhnFGHΔEPSPS. DH5αΔPhnFGHΔEPSPS was Escherichia coli DH5α, which is a CDPS and C-P Lyase-deficient strain, which knocked out the C-P Lyase gene and the EPSPS gene.

It should be noted that DH5αΔPhnFGHΔEPSPS is a defective strain without an antibiotic gene. Most of PhnF, all PhnG and part of PhnH of Escherichia coli DH5α are knocked out with genes related to phosphonates which are degraded by glyphosate. The nucleic acid sequence fragment of the upstream sequence of the 5-terminal end of the FRT DNA fragment and the nucleic acid fragment of the downstream sequence to which the 3 terminus is ligated is shown in SEQ ID NO. Among them, the first to the 318th are the 5th end and the upstream sequence of the PhnF gene of Escherichia coli, the nucleotide sequence of the 319th to the 347th is the FRT fragment, and the 348th to the 1021th are the Escherichia coli PhnH gene 3 The end and its downstream sequence. In addition, most of the EPSPS gene in DH5αΔPhnFGHΔEPSPS was replaced by the FRT fragment, as shown in SEQ ID NO. 12, wherein the first to the 357th positions were the 5-terminal sequence of the E. coli EPSPS gene, and the 358th to the 386th were The FRT fragment, from position 387 to position 818, is the 3 terminal sequence of the E. coli EPSPS gene.

In the second step, the EPSPS and C-P Lyase-deficient strains were obtained as host bacteria in the first step of the example, and the soybean EPSPS gene derived from soybean was introduced into the host strain to obtain a mutant strain, that is, a soybean EPSPS gene mutant library.

The soybean EPSPS gene was cloned into the pADV5 vector by a conventional method, and the pADV5 vector was transformed into the DH5αΔPhnFGHΔEPSPS host strain.

The transformed DH5αΔPhnFGHΔEPSPS was inoculated into MA liquid medium (M9 basal medium + 100 μg/mL Amp), and cultured at 37 ° C, 300 r / min overnight;

The turbid bacterial liquid will be grown and irradiated by radiation mutagenesis, for example, under ultraviolet irradiation for 2-5 min, so that the soybean EPSPS gene is mutated to obtain the corresponding soybean EPSPS mutant gene, that is, the mutant strain, that is, the soybean EPSPS gene mutant library is obtained. Of course, this step can also use chemical mutagenesis treatment to add a chemical mutagen such as EMS or DES to the MA medium to mutate the soybean EPSPS gene.

The third step is screening culture.

5 μL of the above-mentioned bacterial liquid containing the mutant bacteria was added to the screening medium, and the cultivation was continued at 300 r/min and 37 ° C overnight.

The fourth step is sequencing verification.

The monoclonal resistant bacteria grown on the screening medium were selected, isolated, and tested for glyphosate resistance, and sequenced to obtain a sequence of the glyphosate-resistant soybean EPSPS mutant gene.

One of the soybean EPSPS mutant genes is described, and its nucleotide sequence is as shown in SEQ ID NO. 10, and is composed of 1368 bases. The soybean EPSPS mutant gene (designated GmEM gene) is compared with the nucleotide sequence of the wild type soybean EPSPS gene (designated as GmE gene) (as shown in SEQ ID NO. 5) and the amino acid sequence encoded thereby, The result is shown in Figure 4. The soybean EPSPS mutant gene has a base "G" inserted between the 6th and 8th positions from the 5' end to the 3' end, and the base "A" is deleted between the 45th and the 46th position, resulting in the 7th The base to position 44 has undergone a frameshift mutation, and the corresponding amino acid residue sequence encoded by the segment is also mutated from amino acid to carboxy terminus 3 to 15 (as shown in Figure 4); In addition, the 629th base is mutated from "A" to "T", resulting in the amino acid residue 210 of the amino acid residue sequence being mutated from a glutamic acid residue to a proline residue; the 1110th base consists of " A" mutation is "G", and the 1125th base is mutated from "T" to "C", and the base mutations of the two sites do not cause mutation of the corresponding encoded amino acid residue.

Glyphosate resistance test of soybean EPSPS mutant gene was inoculated with Escherichia coli (EPSPS and CP Lyase-deficient strain) transformed with GmEM gene (experimental group) and GmE gene (control group) and contained 0 mM, 1 mM, 5 mM, The growth state of Escherichia coli was observed in a medium of 10 mM, 20 mM, 50 mM, and 10 mM glyphosate. The results are shown in Table 4.

Table 4. Growth saturation index of Escherichia coli transformed with GmEM gene and GmE gene in glyphosate medium containing different concentrations

As can be seen from Table 4, in the medium containing 0 mM glyphosate, the experimental group (including the GmEM gene) and the control group (including the GmE gene) were able to grow normally (saturation index of 4); but in the case of containing 1 mM, 5 mM, 10 mM On the medium with 20 mM glyphosate concentration, the control group could not grow normally, while the experimental group could grow normally (saturation index was 4); on the medium containing 50 mM glyphosate, the control group could not grow normally, and the experiment The group was able to grow vigorously (saturation index was 3); on the medium containing 100 mM glyphosate, neither the experimental group nor the control group could grow normally (the saturation index was 0). This indicates that the soybean EPSPS mutated gene (the nucleotide sequence of which is shown in SEQ ID NO. 10) which is screened in this example is capable of conferring resistance to EPSPS and CP Lyase-deficient Escherichia coli glyphosate, so that Escherichia coli is up to Growth was carried out in a medium of 50 mM glyphosate.

The glyphosate-resistant soybean EPSPS mutant gene screened by the screening method for the glyphosate-resistant mutant gene provided by the present invention has the nucleotide sequence shown in SEQ ID NO. It has resistance to 50 mM glyphosate.

A glyphosate-resistant soybean EPSPS mutant gene (the nucleotide sequence thereof is shown in SEQ ID NO. 10) screened directly by the screening method of the glyphosate-resistant mutant gene provided by the present embodiment of the present invention. Converting soybean or rice or other plants to render the transformed plants resistant to glyphosate.

Example 3

This embodiment provides a defective strain, and specifically, the defective strain is an EPSPS and a C-P Lyase-deficient strain. The EPSPS and C-P Lyase-deficient strains were obtained by knocking out the EPSPS gene and the C-P Lyase gene of Escherichia coli DH5α, TOP10, and BL21 by gene knock-out technique. Specifically, the gene knockout method used in the present example is the same as the gene knockout method used in Example 1 or Example 2.

The EPSPS and C-P Lyase-deficient strains provided in this example can be used in testing the function of the EPSPS gene from the plant of interest. Specifically, the EPSPS and CP Lyase-deficient strains of the present embodiment are used as host bacteria, and the EPSPS gene of the plant of interest is introduced into the host strain, and then the host strain is placed in a basal medium containing no amino acid or protein, that is, restriction Culture in a medium, if normal colony production on the restricted medium is observed, the EPSPS gene from the plant of interest has the function of expressing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase). And EPSPS has normal biological activity.

The EPSPS and C-P Lyase deficient strains provided in this example can also be used in testing the resistance of the EPSPS gene from the plant of interest to glyphosate. Specifically, the EPSPS and CP Lyase-deficient strains of the present example are used as host bacteria, and the EPSPS gene of the target plant is introduced into the host strain, and then the host strain is placed on M9 medium containing different glyphosate concentrations. Culture, for example, growth on M9 medium containing 10 mM, 20 mM, 50 mM glyphosate to test glyphosate resistance of the EPSPS gene from the plant of interest.

The EPSPS and C-P Lyase-deficient strains provided in the present examples can be used in screening for EPSPS mutant genes derived from plants of interest having glyphosate resistance. For a specific method of use, the method for screening a glucagon-resistant EPSPS mutant gene provided in Example 1 or Example 2 can be referred to.

In summary, the screening method provided by the embodiments of the present invention introduces an EPSPS and CP Lyase-deficient strain, and then uses the EPSPS and CP Lyase-deficient strains as host bacteria to introduce an exogenous EPSPS gene from the plant of interest into the defect type. Among the strains, a mutant strain containing the exogenous EPSPS mutant gene, that is, an exogenous EPSPS gene mutant library, was obtained, and an EPSPS mutant gene having glyphosate resistance was screened from the exogenous EPSPS gene mutant library. Since the propagation speed of Escherichia coli is fast and the volume is small, the screening method of the present invention overcomes the problems of long cycle and large area in the existing screening methods, so that the screening method of the present invention has a grass-like selection in the directional screening. The phosphine-resistant EPSPS mutant gene has the characteristics of short cycle, very small footprint, short cycle and simple operation. Moreover, the screening method of the present invention uses EPSPS and CP Lyase-deficient Escherichia coli as host bacteria, and effectively excretes the mutation of the EPSPS gene and the CP Lyase gene of the host bacteria to produce glyphosate resistance, so that the selected The results are more scientific and reliable. Screening out the genetic mutations of the EPSPS gene from plants can greatly improve the screening speed and time. Usually, it takes only 1-2 weeks to complete the screening work, and obtain the glyphosate-resistant mutant gene to reduce the screening work. cost. In addition, the glyphosate-resistant mutant gene obtained by the screening method provided by the present invention can be reused for transforming the corresponding plant species, overcoming the bottleneck phenomenon of most of the current resistance genes that can only be transferred from microorganisms to crops. It will help to eliminate public prejudice against GM plants and promote the development and promotion of GM technology. In addition, the EPSPS and CP Lyase-deficient strains provided by the present invention can be applied in testing the function of the EPSPS gene of the plant, and can also be applied in testing the resistance of the plant EPSPS gene to glyphosate, and the application thereof is convenient. The result is more scientific and reliable.

The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims

A method for screening glyphosate resistant genes, comprising:

The interfering gene of the source strain is knocked out by a knockout technique to obtain a defective strain derived from one of Escherichia coli DH5α, TOP10 and BL21, and the interference gene includes an EPSPS gene and a CP Lyase gene, the defect The strain is an EPSPS and CP Lyase-deficient strain; the exogenous EPSPS gene is first introduced into the defective strain, and then subjected to mutagenesis treatment to obtain a first mutant strain containing an exogenous EPSPS mutant gene, the exogenous EPSPS The gene is from the plant of interest;

Or the exogenous EPSPS gene is first mutated to obtain an exogenous EPSPS mutated gene, and the exogenous EPSPS mutated gene is introduced into the defective strain to obtain a second mutant bacterium;

The first mutant strain or the second mutant strain is placed on a screening medium containing glyphosate for screening culture to obtain a monoclonal resistant strain having glyphosate resistance;

The monoclonal resistant bacteria were subjected to sequencing verification to obtain an EPSPS mutant gene having glyphosate resistance.
The glyphosate resistance gene screening method according to claim 1, wherein the mutagenesis treatment is a chemical mutagenesis treatment or a radiation mutagenesis treatment.
The glyphosate resistance gene screening method according to claim 1, wherein the mutation treatment is performed by using the exogenous EPSPS gene as a template, and PCR is performed by a mismatch PCR method or a DNA Shuffling method to obtain the exogenous EPSPS mutation. gene.
The method for screening glyphosate resistant genes according to claim 1, wherein the plant of interest is rice, soybean, wheat, corn, barley, sorghum, tobacco, cotton, sweet potato, poplar, potato, cabbage, kale or green pepper. .
A glyphosate-resistant EPSPS mutant gene screened by the glyphosate-resistant gene screening method according to any one of claims 1 to 4.
The use of an EPSPS mutant gene according to claim 5 for transforming a plant to confer resistance to the plant to the glyphosate.
A defective strain obtained by knocking out an interfering gene by a source strain by using a gene knockout technique, the interference gene comprising an EPSPS gene and a CP Lyase gene, the source strain being selected from the group consisting of Escherichia coli DH5α, TOP10 And one of BL21, the defective strain being an EPSPS and CP Lyase-deficient strain.
Use of the defective strain according to claim 7 for testing the function of an EPSPS gene derived from a plant of interest.
Use of the defective strain according to claim 7 for testing resistance of an EPSPS gene from a plant of interest to glyphosate.
Use of the defective strain according to claim 7 for screening an EPSPS mutant gene derived from a plant having glyphosate resistance.